This application addresses broad Challenge Area (06) "Enabling Technologies" and specific challenge topic 06-AG-101* Neuroscience Blueprint: Development of non-invasive imaging approaches or technologies that directly assess neural activity. It also applies to specific challenge topics 06-NS-101 (Developing minimally invasive measures of neural activity) and 06-NS-103 (Breakthrough technologies for neuroscience). One of the greatest challenges for neuroscience in the 21st century is to understand how the billions of neurons that form the brain communicate with one another to produce complex behaviors. The ultimate benefit from this type of research will come from deciphering how dysfunctional patterns of activity amongst neurons lead to devastating symptoms in a variety of neuropsychiatric disorders. Unfortunately, little is known regarding how neural computations in the brain interpret sensory inputs or generate behaviorally relevant responses. This is due in part to the current lack of tools to interrogate the activity of large numbers of neurons in the intact brain. Through an interdisciplinary research collaboration between physicists and neuroscientists, we have developed a high-speed 2-photon microscope for calcium imaging that combines fast resonant scanning mirrors and multi-beam imaging to achieve image acquisition rates more than 2 orders of magnitude faster than conventional 2-photon microscopes. To avoid the fundamental limitation of scattering ambiguity with multiple beams in deep-tissue 2-photon microscopy, we propose an innovative approach to detect and resolve scattered fluorescence emission from separate beams at different times. Specifically, we split the laser beam into four beam lets and then delay each beam optically from the others by 3 ns. We call this method Spatio- Temporal Excitation-emission Multiplexing (STEM). The signals from all four beams are detected by a state- of-the-art GHz bandwidth photodetector. Our microscope therefore preserves the unique advantages of 2- photon microscopy, including its ability to excite fluorophores deeper in the tissue, its reduced photo damage and its exquisite spatial resolution. We now propose to systematically optimize our STEM microscope in order to achieve fluorescence lifetime imaging (FLIM) capability and 4-color imaging. The ultimate goal is to achieve unprecedented 6-D (x, y, z, t, [unreadable], ") bio-imaging at the single cell level. In addition, we propose a series of in vivo calcium experiments to systematically dissect the micro-scale connectivity of neocortical circuits. First, we will calibrate our STEM system to demonstrate its superior action potential detection compared to conventional 2-photon calcium imaging. Next, we will examine the spatiotemporal dynamics of large ensembles of layer 2 and layer 3 neurons in barrel cortex in response to whisker deflections, by recording from hundreds of these neurons simultaneously in 2-D and 3-D at unprecedented speeds. Within 2 years, the instrument will be optimized and we will be able to characterize, for the first time, the functional wiring diagram of entire complement of neurons within a volume of neocortex. PUBLIC HEALTH RELEVANCE: We have recently developed a high-speed microscope to record the activity of neurons in the intact brain non-invasively. The goal of the proposed challenge grant is to optimize this instrument and then use it to investigate how brain circuits are assembled during development in areas important for emotion, cognition and creativity, as well as for learning and memory. This innovative tool will allow neuroscientists to design experiments that can generate new ideas regarding how subtle alterations in brain wiring could result in devastating neuropsychiatric disorders such as schizophrenia, autism, mental retardation or bipolar disorder.